We are proposing to develop a new strategy to overcome radiation resistance in B- lineage acute lymphoblastic leukemia (ALL) using C-61, a novel SYK kinase substrate binding (P)-site inhibitor, for targeting and disrupting the anti-apoptotic SYK-STAT3 signaling network in leukemic B-cell precursors. It is our central working hypothesis that the treatment outcome of relapsed B-lineage ALL patients can be improved by inhibition of the SYK-STAT3 molecular target. This would be accomplished by using C-61 in combination with total body irradiation (TBI) in the context of HSCT. Under Specific Aim 1, we will examine the effects of the SYK P-site inhibitor C-61 on in vitro radiation resistance of primary ALL cells from relapsed B-lineage ALL patients using quantitative flow cytometric apoptosis assays and clonogenic assays. We hypothesize that C- 61 will markedly enhance radiation-induced apoptosis of primary B-lineage ALL cells and augment radiation- induced death of their clonogenic fraction by increasing their radiation sensitivity and impairing their capacity to repair sublethal radiation damage. Under Specific Aim 2, we will examine the effects of C-61 on in vivo radiation resistance of primary ALL cells from relapsed B-lineage ALL patients using SCID mouse xenograft models of relapsed B-lineage ALL and sublethal total body irradiation (TBI). Our hypothesis is that C-61 plus TBI regimens will be more effective than TBI alone in improving the event-free survival outcome of SCID mice challenged with primary B-lineage ALL cells. Under Specific Aim 3, we will examine the association between the kinase expression profiles of primary ALL cells from relapsed B-lineage ALL patients and their in vitro as well as in vivo radiation resistance. In an effort aimed at identifying a composite biomarker profile that will help select patients most likely to benefit from C-61, we will correlate the kinase protein expression and activity levels of SYK, BTK, and JAK kinases of primary B-lineage ALL cells with their radiation resistance, sensitivity to C-61 induced radiosensitization in vitro, as well as C-61 induced potentiation of the anti-leukemic potency of sublethal TBI in vivo. Under Specific Aim 4, we will study the efficacy and safety of C-61 containing single dose TBI regimens at both sublethal (2 Gy) as well as clinically applied (7 Gy) total radiation dose levels in a syngeneic murine HSCT model of radiation-resistant BCL-1 murine B-lineage leukemia. We will evaluate the efficacy and safety of TBI at doses ranging from 2-10 Gy in combination with C-61 in BALB/c mice inoculated with 1x106 BCL-1 cells in the context of syngeneic BMT. We hypothesize that the addition of C-61 will not increase the non-hematologic toxicity of TBI, while markedly potentiating its anti-leukemic efficacy. We anticipate that the incorporation of C-61 into the pre-HSCT TBI regimens of patients with relapsed B-lineage ALL will improve their treatment response and survival outcome. The proposed research has the potential provide the foundation for the development of paradigm-shifting HSCT strategies that employ C-61 containing novel TBI regimens.